The major surface antigen of the hepatitis B virus is a 25 kDa protein, HBsAg. The protein is known in three forms: preS1, preS2 and S. The preS1 and preS2 forms include 14 and 39 amino acids that are cleaved from their N-termini in vivo to yield the 226 amino acid S form. (Valenzuela P., et al. (1979) Nature, 280:815; Valenzuela P., et al., Synthesis and assembly of hepatitis B virus surface antigen particles in yeast. Nature. (1982) 298:347–350. Miyanohara A., et al., Expression of hepatitis B surface antigen gene in yeast. PNAS U S A (1983) 80(1):1–5)
Over the past two decades, recombinant hepatitis B surface antigen expressed as the S form in yeast cells (rHBsAg) has superseded plasma-derived antigen as a vaccine against hepatitis B infection (Valenzuela et al., 1982; McAleer et al., 1984). In the plasma of infected animals, the surface antigen protein assembles into 22 nm particles comprising lipids and HBsAg. However, the assembly of the yeast produced rHBsAg into these virus-like particles has remained poorly understood.
It has been established that reduction of the disulfide bonds in HBsAg abolishes or greatly decreases both its antigenic and immunogenic properties (Reviewed by Tiollais et al., 1981, Guesser et al., 1988; Mishiro et al., 1980; Chen et al, 1996; Hauser, et al 1988). For example, eleven out of the total 14 cysteine residues are conserved among three different type of S protein of Hepatitis B viruses, namely, HBsAg of human, woodchuck, and ground squirrel viruses. However, these three types of S protein share only medium to low overall sequence homology among (Stirk et al, 1992). Interestingly, all 8 cysteine residues in the“a” determinant loop (62 aa between predicted Helix B and Helix C) are fully conserved. This indicates that disulfide bonds may have an important role in maintaining the structural integrity of the antigenic determinants or epitopes (Stirk et al., 1992).
The importance of the presence of disulfide bonds to the functional and structural integrity of proteins has been well documented, particularly for ribonuclease A (Ubuka T., (1996) Protein disulfide isomerase-catalyzed renaturation of ribonuclease A modified by S-thiolation with glutathione and cysteine. Biochem Mol Biol Int. 38(6):1103–10; Fahey, R. C., (1977) Biologically important thiol-disulfide reactions and the role of cyst(e)ine in proteins: an evolutionary perspective. Adv Exp Med Biol. 86A:1–30; Lyles M M, et al., (1991) Catalysis of the oxidative folding of ribonuclease A by protein disulfide isomerase: dependence of the rate on the composition of the redox buffer. Biochemistry. 22;30(3):613–9).
In the case of rHBsAg, the correct disulfide bond pairings are important antigenic determining factors since they are most likely required for the integrity and stability of the major epitopes. (Wampler et al., 1985). Multiple chemical forms of hepatitis B surface antigen. Proc. Nat. Acad Sci. 82:6830–6834. On over-expression in yeast cells, it is believed that the HBsAg molecules need to find a lipid environment for folding into the membrane-embedded structures as seen in the plasma-derived 22 nm lipid/protein particles. However, because of a lack of the control of the cellular redox environments, unlike the propagation of the virus in the infected cells, during over-expression, and sub-optimal conditions during purification and formulation of rHBsAg, the recombinant proteins are expected to assume certain sub-optimal conformations as a result of mismatched disulfide bond pairings.
Although some cysteine-rich proteins or peptides are reported to have strong propensity to form the correct disulfide bond pairings during oxidative refolding (Moroder et al., 1996; Mosiol et al., 1994), productive folding is always in competition with nonproductive folding. These latter pathways lead to either wrong disulfide bond pairings or aggregation of polypeptides.
The rate and yield of oxidative renaturation of small reduced polypeptides have been reported to be influenced by the ratio of low molecular weight disulfide/thiol compounds. The relationships are rather complex and do not parallel the redox potential of the system, as reported using reduced RNAse A as a model protein (Wetlaufer et al., 1987).
It is reported that a redox buffer with both forms of glutathione, i.e., GSSG/GSH mixture, might facilitate the conformational searching by promoting the formation of the correct pairings and unlocking the mismatched ones. However, the use of GSSG/GSH mixtures was only demonstrated for some small proteins and peptides (Moroder, et al., 1996).
It is believed that in a natural system of cells infected by HBV, the nascent HBsAg molecules need to find a lipid environment for the formation of certain intramolecular and/or intermolecular disulfide bonds that lead to proper folding into the native conformation of the protein. In the natural system, this process leads to the formation of particles of approximately 22 nm in diameter made up predominantly of HBsAg protein associated with a lipid membrane. Similarly, when expressed in yeast cells or other expression host, it is believed that the nascent rHBsAg needs to find a lipid environment prior to the spontaneous folding into membrane-embedded structures.
However, in recombinant processes, the rHBsAg is over-expressed in a non-natural system using a host cell. Insect, yeast and CHO cells are commonly used although other cell types may be employed. When overproduced in such a system, the rHBsAg is an assortment of aggregations of scrambled forms and non-native conformations due to mismatched disulfide bond pairings. These artifacts of over-expression yield molecules locked into conformations of low antigenicity. Therefore, once produced, the over-expressed rHBsAg is typically processed outside a cellular environment to eliminate some of the undesired artifacts of over-expression.
In a method presently used in the art, once the over-expressed rHBsAg is purified from the host cells, the antigen is treated with thiocyanate in an oxidative step to induce a conformational search and yield the form of rHBsAg known as Form III (Wampler, et al., 1985) (See FIG. 1). Thereafter, formalin treatment is used to lock the rHBsAg into whatever conformation it has assumed under the chaotropic, partially denaturing conditions of the thiocyanate treatment. Finally, the rHBsAg it is precipitated with adjuvant.
Previous studies reported that one could achieve an enhancement of antigenicity of rHBsAg by incubation at elevated temperatures as well as the inhibitory effect of formalin to the same process. Moreover, the role of the different disulfides being generated (i.e., intra- and intermolecular) was also described (Wampler, et al., (1985)). In these studies, the conformational search for the thermodynamically most stable forms of rHBsAg occurred spontaneously. Therefore, the optimal percentage of correct three-dimensional structures of the rHBsAg could not be obtained by those methods. Most importantly, the reported procedures yield product that varies for potency as well as consistency. This is possibly due to the poorly-controlled redox conditions, residual metals and surface contacts during process and formulation. Thus, vaccines including rHBsAg made by previously reported methods varies considerably in amount of protein required to induce a protective response in a vaccinated subject.